An Introduction to Neurochemistry: Acetylcholinesterase Assay

adapted from material presented at a PKAL conference on Neurobiology 1997

The ability of a neuron to communicate with other neurons through a chemical

synapse is a source of endless fascination for neuroscientists. The chemical vehicles
that enable this communication are neurotransmitters. Neurotransmitters can be
categorized into four groups monoamines, amino acids, peptides and acetylcholine.
They are synthesized, packaged and transported (if necessary) to the terminal of the
presynaptic cell. The arrival of the action potential at the terminal of this cell triggers
the release of neurotransmitter into the synaptic cleft. After diffusion across the
synapse, the neurotransmitter can activate receptors on the postsynaptic cell resulting
in excitation or inhibition of that cell. Finally, the neurotransmitter is inactivated through
enzymatic breakdown, re-uptake or by it's diffusion. Analysis of the various
biochemical events associated with synaptic transmission is the domain of the
neurochemist. This lab exercise concentrates on the neurotransmitter acetylcholine
(ACh) and the enzyme which breaks it downs acetylcholinesterase (AChE), as an
introduction to neurochemical methods.

What you need to know..........

Acetylcholine (ACh) is one of the major neurotransmitters of the brain. It was first
described in the PNS in a classic experiment by Otto Loewi (1921). While stimulating
the vagus nerve of a frog to slow down its heart, Loewi collected the perfusate and
applied it to the heart of a second frog. This heart also slowed down! Loewi and his
colleagues, "the first neurochemists", demonstrated that this inhibitory substance in
the perfusate was mimicked in every way by ACh. Then in 1936, H. H. Dale
demonstrated that ACh is the neurotransmitter that has an excitatory effect in the
neuromuscular synapse. But how can ACh be inhibitory in the heart yet excitatory in
the muscle? It is now known that the effect of a neurotransmitter is not determined by
the neurotransmitter itself, but by its postsynaptic receptor. For example, the nicotinic
ACh receptor is an ionotropic receptor and a postsynaptic nerve will respond
differently than one containing the muscarinic ACh receptor (a metabotropic receptor).

The function of ACh in the PNS is well documented in the autonomic nervous system
and the neuromuscular junction. The autoimmune disease myasthenia gravis is
thought to be due to a lack of cholinergic receptors in the PNS. You can read more
about this disease in your book (Chapter 17). The function of ACh in the brain is less
well defined, but ACh is thought to be related to motor activity, learning and memory,
and controlling sleep stages. Deficits of ACh are associated with Alzheimer's and
Huntington's diseases. Figure 1 shows some of the regions in the brain where cell
bodies and axonal projections that use cholinergic neurons are found.

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Figure 1. Above is a map of the acetylcholine pathways in the rat brain. These pathways help
regulate global functions that rely upon the cerebral cortex; such functions include attention, arousal,
motivation, memory and consciousness. You should observe where these reside in the brain. The
details of the different types of connections are beyond the scope of this lab. The basal forebrain
contains two groups of cholinergic neurons: (1) the medial septal group (medial septal nucleus and
vertical diagonal band) that project cholinergic axons to the hippocampus and parahippocampal
gyrus and (2) the nucleus basalis group (yellow and orange; nucleus basalis, substantia innominata
(not shown) and horizontal diagonal band) that project cholinergic axons to all parts of the neocortex,
parts of limbic cortex and to the amygdala. The cholinergic pontomesencephalon neurons (blue;
laterodorsal tegmental and pedunculopontine tegmental nuclei ) project onto hindbrain, thalamus,
hypothalamus and basal forebrain. The red symbols indicate ACh cell bodies and projections (Text
adapted from the web site of Dr. Nancy Woolfe .(http://www.bol.ucla.edu/~nwoolf/).

Acetylcholinesterase (AChE) is an important enzyme which regulates the effects of

acetylcholine at cholinergic synapses. AChE's main function is to terminate the effects
of ACh after it is released, acting like an off switch. AChE is one of the most efficient
enzymes in existence, having a turnover time of 150 usec, which is equivalent to the
hydrolysis of 5,000 ACh molecules/molecule of enzyme/sec. It is predominantly found
bound to the postsynaptic membrane. AChE has two sites which bind to the cationic
and esteric domains of ACh, cleaving the molecule between these sites (see Fig. 2).

Biochemistry: ACh is synthesized in neuron terminals in a reaction catalyzed by the

enzyme choline acetyltransferase (ChAT) (see Figure 2 below). The choline is derived
from membrane phospholipids while the acetyl CoA is a breakdown product of
glucose. ACh is degraded into choline and acetate by acetyIcholinesterase (AChE).
The choline is taken back into the presynaptic cell and reused to produce additional
ACh (see Figure 3 for how ACh is metabolized).

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 ChAT
acetyl CoA + choline =========> acetylcholine + CoA

AChE
Acetylcholine <==========> choline + acetate

Figure 2. The structure of acetylcholine. The reaction that produces ACh is catalyzed by choline
acetyltransferase (ChAT) whereas the breakdown of ACh is catalyzed by acetyIcholinesterase (AChE)
The arrow indicates where ACh is cleaved AChE at the ester linkage.

Measuring AChE by the methods described in this lab exercise is a simple and quick
way to look at ACh distribution, however, it is still unclear whether this enzyme
consistently co-localizes with ACh. So why not measure ACh itself as a marker for the
cholinergic system? ACh degrades rapidly after death and it is very difficult to sacrifice
an animal quickly enough to preserve the acetylcholine.

Figure 3. Acetylcholine metabolism. Note that acetyl CoA is primarily found in the mitochondria but
that the synthesis of ACh is in the cytoplasm. It is unknown how the transport of acetylcholine out of
the mitochondria occurs. Choline can be synthesized by the brain but it is thought to actually arrive in
the brain in either a phospholipid form (phosphatidylcholine) or as free choline (adapted from
Biochemical Basis of Neuropharmocology).

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Enzyme Kinetics: The kinetics of enzyme/substrate interactions can be displayed in a
variety of ways: rate of reaction against time; rate of reaction against enzyme
concentration; rate of reaction against substrate condensation The shape of all these
curves is similar since they all start off linearly and then level off because either the
substrate or the enzyme becomes limiting. The effect of substrate concentration on
the reaction rate is plotted in Fig. 4. This relationship is described mathematically in
the Michaelis-Menten equation (see Fig. 4). Note that there are two constants derived
from this curve: Vmax which is the maximum velocity of the reaction, and Km which Is
the substrate concentration at which half the maximum rate (1/2 Vmax) is achieved.
These two constants are referred to frequently when dealing with enzyme kinetics.

Enzymes can be inhibited by a substance which can bind to the active site. This
inhibitor will prevent the binding of the neurotransmitter (substrate); however if an
excess of neurotransmitter is present, it can out compete the inhibitor and the enzyme
will become active again. This is known as competitive inhibition. An inhibitor can also
bind elsewhere on the enzyme and change its shape so that the active site is no
longer available This inhibition is not effected by the concentration of substrate and is
called noncompetitive inhibition. These two different kinds of inhibition can be
compared mathematically by using the Lineweaver-Burke equation (which is derived
from the Michaelis-Menten equation). Based on what you have had in Bio220, you
should be able to diagram what a graph of the reaction with a competitive and a
noncompetitive inhibitor would look like.

Figure 4. Plot of reaction rate versus increasing substrate concentration. From such a graph
you can calculate Vmax and K m as indicated.

A variety of compounds have been found that inhibit AChE. Nerve gases, a class of
organophosphorus compounds, such as sarin, are poisons which bind irreversibly to
AChE and can have fatal consequences. The drug Cognex (tacrine hydrochloride) is a
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drug used to treat patients with Alzheimer's disease. Tacrine and other AChE
inhibitors prevent the degradation of ACh and thus prolong its action (see Figure 6 for
sites of action of drugs that act on cholinergic synapes).

Figure 5. Sites of action of various drugs at cholinergic synapses.

Site 1: ACh synthesis is blocked by styryl pyridine derivatives such as NVP.
Site 2: Transport of ACh into vesicles is blocked by vesamicol (AH5183).
Site 3: Vesicle release is promoted by β-bungarotoxin, black widow spider venom, an La3+ . It
is blocked by botulinum toxin, cytochalisin B, collagenase pretreatment and Mg2+ .
Site 4: The ACh receptors are activated by cholinometic drugs and anticholinesterases.
Nicotinic receptors are blocked by rabies virus, curare, hexamethonium, or
dihydroerythroidine. Agonists include n-methylcarbamylcholine, and dimethylphenyl
piperazinium. Muscarinic receptors are blocked by atropine, pirenzepine, and quinuclidinyl
benzilate.
Site 5: Muscarinic receptors found in the presynaptic membrane may be blocked by AFDX-
116, atropine, or quinuclidinyl benzilate.
Site 6: AChE is inhibited by physostigmine (eserine), sarine, tacrine, DFP, or soman.
Site 7: Choline uptake blockers include hemicholinium-3, troxypyrrolium tosylate, or AF64A.
(adapted from Biochemical Basis of Neuropharmocology)

In this lab..........

This experiment describes a method for measuring the activity of AChE in different
regions of the rat brain. The data can then be compared with AChE stained brain
sections. The effect of the inhibitor tacrine will be examined to determine whether it is
a competitive or a noncompetitive inhibitor.

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THE PROTOCOL

The activity of AChE will be measured according to a method developed by Ellman et

al. 1961. This method employs acetylthiocholine iodide (ATChI) as a synthetic
substrate for ACh E. ATChI is broken down to thiocholine and acetate by AChE and
thiocholine is reacted with dithiobisnitrobenzoate (DTNB) to produce a yellow color.
The quantity of yellow color which develops over time is a measure of the activity of
AChE and can be measured using a spectrophotometer (spec. 20).

These coupled reasons are represented by the following equations:

AChE
acetylthiocholine iodide ==============> thiocholine + acetate

thiocholine + dithiobisnitrobenzoate ======> yellow colored products*

* produce of the reaction are 2-nitrobenzoate-5 mercaptothiocholine and 5-thio-2-nitrobenzoate (the latter is the
yellow product)

The activity of an enzyme is generally expressed as a rate: the quantity of substrate (in
moles) which is broken down by a known amount of enzyme per unit time. In this
case, it will be the amount of ATChI which is broken down by AChE per minute.

1. Equipment and Solutions

A. Equipment
spectrophotometer scales
single edged razor blades kimwipes
vortex pipettes - 5 ml
ice buckets and ice Pipetmen - various sizes
rubber gloves capped 25 ml (centrifuge) tubes
clean or disposable 13 x 100 mm glass test tubes and test tube rack
aluminum foil stop watches
sonicator or homogenizer Rat Atlas

B. Solutions
0.1M phosphate buffer (PB), pH 8.0
0 075M acetylthiocholine iodide (ATCld)
0.01M dithiobisnitrobenzoate (DTNB)

You will prepare a brain homogenate and dilute it to approximately @5 mg/ml. The solutions, 0.1M
phosphate buffer (PB), pH 8.0 0.01M dithiobisnitrobenzoate (DTNB) and a stock of acetylthiocholine
iodide (ATChI) at 0.1M will be provided. You will need to use the ATChI at the following
concentrations: 0.1M; 0.075M; 0.05M: 0.025M; 0.01M and 0.005M. You may find it useful to
calculate how you will make the diluted ATChI solution prior to coming to class.

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II. AChE assay in Different Brain Regions:
A. Prepare brain homogenates

TIP; Keep all tubes and solutions on ice during the experiment.

1. Weigh a sample of cortex (each student needs ~ 30 mg) and place it in a

labeled tube. Add 1 ml of PB/30 mg tissue (30 mg/ml). Briefly sonicate or
homogenize this solution until the brain is uniformly dispersed in the buffer.
Place the tube on ice.

2. Prepare the brain samples from cerebellum, striatum and hippocampus (as
in #1 above). To help you identify these regions, refer to Figures 6 & 7. Clearly
label the homogenates and store them on ice.

B. Assay (construct a flow sheet to help you stay organized flow sheet):

1. Turn on the spectrophotometer and set at 412 nm. Let it warm up for at least
15 minutes prior to reading.

2. Label the assay tubes - four tubes (3 for the assay and one for a control) for
each brain region: cortex, cerebellum, hippocampus and stratum (4 x 4 = 16).

3. Pipette 3 ml PB into each assay tube

4. Using a pipette add 200 uL of cortex homogenate to each of the four labeled
assay tubes. Vortex each tube and return it to the ice. Repeat this step for the
other three brain regions.

TIP: To ensure accurate results, remember to use good pipetting technique. If

you are not familiar with automatic pipetters, check with your instructor.

5. Zero the spec. 20 without a tube by setting the needle to O transmittance

(use knob on the left front).

6. Add 100 uL DTNB to the first cortex tube, vortex, and place it in a test tube
rack for five minutes. This allows the solution to reach room temperature.

TIP: To save time, 100 uL of DTNB can be added to the next tube which can be
stabilizing at room temperature while the protocol is followed for the first tube.

7. Vortex and quickly wipe the outside of the tube with a kimwipe (to remove
moisture and fingerprints which could interfere with the passage of light). Place
the tube In the spec. 20 and zero the spectrophotometer to 0 absorbance using
knob on right) This will be your baseline reading before measuring product
formation.
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Figure 6. The rat brain. a - lateral aspect; b - ventral aspect; c - midsagittal view; and d - dorsal
aspect of the brain stem. Refer to Figure 8 for a dissection plan. (Figure adapted from Experimental
Psychobiology: A laboratory manual, edited by Benjamin L. Hart, W. H. Freeman and Company 1976)

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Figure 7. Dissection of the cerebral hemispheres to the rat brain. a. You will need to collect a
cerebellum sample by cutting the cerebellar peduncles to remove the cerebellum; b . You will also
need to collect a cerebral cortex sample which is a thin layer that should be gently pulled away. The
appearance of the brain with the cerebellum and the cerebral cortex removed is shown; c. Gently
remove the corpus callosum to get at the underlying structures. The appearance of the brain with the
corpus callosum removed; d. You will need to collect the striatum sample which we can do by taking
the caudate-putamen nucleus (these comprise the striatum). Remove this portion. Next you can
remove the hippocampus. You will need to cut it away from the fornix. Shown is removal of the
hippocampus after the connections to the fornix have been cut. (Figure adapted from Experimental
Psychobiology: A laboratory manual, edited by Benjamin L. Hart W. H. Freeman and Company 1976)

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 8. Take the tube out of the spectrophotometer, quickly add 20 uL ATChl and
vortex.

9. Immediately return the tube to the spec. 20. Note the time and take a zero
reading of absorbance. Take readings at 30 sec, 60 sec, 2 min., and 3 min.
and record the data in a table.

10. Repeat this procedure (steps 6-10) for the other 2 cortex l homogenates.
Run the control through the same procedure except do not add substrate
(ATChI) but add 20 uL PB instead.

11. Assay the 3 cerebellum samples, the 3 striatum samples and the 3
hippocampus samples and their controls.

TIP: Striatum rates may be very fast and go off the scale of the spec. 20. If this
happens, dilute these samples by 2 but remember to double the rates when
doing the calculations.

C Calculate of the rate of the reaction:

1. Graph the data for the different brain regions - change in absorbance/min.
against time. Are the graphs linear?

2. Calculate the rate of color change per minute for each reading and average
the rates within each three minute run. Then average the rates between each
run for each brain region, calculate the rate of the reaction according to the
following equation:

R = A/(1.36*10 4) x 1/(200/3320)C o = 1.22(10 -3) A/Co

R = rate, in moles substrate hydrolyzed/min. g tissue

∆A = change in absorbance/min.

Co = original concentration of tissue (mg/ml)

200/3320 are volume corrections

1.36 (10 4) is the extinction coefficient of the yellow product

3. Make a bar graph to show the enzyme activity of each brain region.

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 III. Comparison of the measurements of AChE distribution with AChE stained
slides (pictures)
It is possible to dye thin sections of brain with a stain which is specific for
AChE. A rat atlas has photographs of sections of rat brain stained for AChE in
addition to sections labeled with the Nissl stain (a stain for cell bodies). It is
interesting to compare these stained sections with the results from the
preceding protocol concerning concentration of AChE in different brain regions.
The data should be complementary.

1. Look carefully at the AChE stained sections and draw representative

sections from each slide. Make sure to label the brain structures and indicate
how they are stained. Note in the drawing which areas of the brain contain
relatively more and relatively less AChE.

2. Compare the distribution of AChE as determined lay staining patterns in the

sections with your estimates of AChE distribution from the neurochemical
method.

WHERE DO YOU GO FROM HERE?

Collect and organize your data in a lab report. The following questions should help
you focus on the meaning of your data:

1. Are your calculated reaction rates linear? If not, can you explain what is happening?

2. Does the distribution of AChE determined by the Ellman method correlate to the
distribution of AChE on the stained slides (or in the atlas)?

3. Were you pleased with how you designed your experiment? Can you think of ways
to improve it?

POINTS TO PONDER

1. From your findings in this lab, is it possible to relate the patterns of enzyme
distribution in different structures of the brain to functions associated with these
structures?

2. It is important to consider whether the concentration of an enzyme such as AChE is

necessarily indicative of the concentration of the neurotransmitter ACh. Assuming that
you have the knowledge and skills to measure ACh directly and there is a poor
correlation between the concentration of ACh and AChE, can you think of a reason why
this should be so?

3. If you were designing a drug to enhance ACh levels, would you want to use a
competitive or a noncompetitive inhibitor? Is this an important feature to be
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considered when designing a drug?

4. Consider a neurological disorder that is thought to involve the cholinergic system.

Could tacrine be used to treat the condition? Suggest other chemicals that could be
used to treat the disorder and where they would have their effect in the life cycle of The
neurotransmitter.

REFERENCES

Cooper, J.R., F.E. Bloom, and R.H. Roth. 1992. The Biochemical Basis of
Neuropharmacology, 6th ed. Oxford University Press, New York, NY.

Dale, H. H., W. Feldberg, and M. Vogt. 1936. Release of acetylcholine at voluntary

motor nerve endings. J. Physiol., 86- 353-380.

Ellman, C.L., D. Courtney, V. Andres, and R. Featherstone. 1961. A new and rapid
colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol., 7:
8895.

Fonnum, F. 1975. A rapid radiochemical method for determination of

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Greenfield, S. 1983. Acetylcholinesterase may have novel functions in the brain. TlNS,
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Hardy, J., L Heimer, R. Switzer, and D. Watkins. 1976. Simultaneous demonstration of

horseradish peroxidase and acetylcholinesterase. Neuroscience, 3: 1-5.

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Koelle, G. B. 1955. Histochemical identification of acetylcho]inesterase in cholinergic,

adrenergic and sensory neurons. J. PharmRcol. and Exper. Ther., 114: 167-184.

Lexrey, A. I., B. H. Wainez, E.J. Mufson and M. M. Mesulam. 1983. Co-localization of

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Loewi, O. 1921. Pfügers. Arch., 189: 239;193: 201.

Paxinos, G. and C. Watson. 1986. The Rat Brain in Stereotaxic Coordinates, 2nd ed.
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Quinn, D. M. 1987. Acetylcholinesterase: Enzyme structure, reaction dynamics, and
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Robertson, R.T., C. F. Holunann, J.L. Bruce and J.T. C:oyle. 1988. Neonatal
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